Non-invasive assessment of perfusion defects with myocardial contrast echocardiography can potentially be a valuable tool in the evaluation of patients with coronary artery disease. However, quantification of under-perfused myocardium with two-dimensional echocardiography has intrinsic limitations due to the restricted number of standard imaging planes. Real-time three-dimensional echocardiography (RT3D echo) is a novel technique that provides volumetric images without ECG or respiratory gating. The aim of this study was to validate the use of RT3D echo for the quantitative assessment of myocardial perfusion defects in an in vivo model of acute coronary occlusion. To this end, 20 sheep underwent acute ligation of the left anterior descending (n=14) or the posterior branch of the circumflex (n=6) artery under general anesthesia. RT3D echo images were obtained after coronary occlusion and left atrial injection of a contrast agent (perflenapent emulsion or Echogen?; 0.8-1 ml). After image acquisition, Evans blue dye was injected into the occluded coronary artery for subsequent anatomic identification of under-perfused myocardium. The mass of the entire left ventricle (LV) and of the under-perfused myocardial region were directly measured following euthanasia. Blinded off-line calculation of LV mass and perfusion defect mass from RT3D echo images were performed using an interactive aided manual tracing technique based on a Silicon Graphics workstation with customized software. Total LV mass ranged from 68 to 141 g (mean+/-SD: 92+/-24). The mass of the perfusion defect ranged from 0 to 43 g (mean+/-SD: 16+/-9) or 0 to 36% of total LV mass (mean+/-SD: 18+/-9%). RT3D echo estimation of total LV mass strongly correlated with the anatomic measurement (r=0.91; y=- 2.54+1.04x; SEE= 11.9 g). RT3D echo calculation of the mass of under- perfused myocardium also strongly correlated with the anatomic measurement, both in absolute (r=0.96; y= 2.01+0.87x; SEE= 2.2g) and percent (r=0.96; y= 0.11+1.02x; SEE= 2.8%) terms. Thus, RT3D echo with myocardial contrast opacification accurately predicts the amount of under-perfused myocardium in an animal model of acute coronary occlusion. This technique may therefore be useful for the quantitative assessment of myocardial perfusion defects in patients with coronary artery disease. - three-dimensional echocardiography; myocardial perfusion; coronary circulation